Spatio-temporal fluctuations of Ca2+ are known to occur in the signaling pathways that regulate fertilization, exocytosis, endocytosis and cell motility, while dysfunctional regulation of Ca2+- regulated signaling pathways is linked to arrhythmic contraction of cardiac and skeletal muscle. While it is possible to image naturally-generated spatio-temporal waveforms of Ca2+ and signaling proteins, it has proven to be extraordinarily difficult to generate artificial spatio- temporal waveforms of Ca2+ and its effecto proteins in cells. This capability would allow us to gain a better understanding of the mechanisms that give rise to frequency-encoded molecular signaling that occur during growth factor-triggered generation of endosomal waves in HeLa cells, and to assess how the propagation and interferences of Ca2+ waves affect the behavior of properties of individual cells within an epithelium. The new photochromic chelators described in this proposal are unique in that they undergo rapid and reversible, orthogonal, visible light-driven transitions between two chemically and functionally distinct meta-stable states. The Ca2+-affinities of the two states of our BAPTA-based chelator, which integrate a red-shifted azobenzene, benzspiropran or spiro-amidorhodamine actuator, differ by more than 2 log units of concentration. The populations of the high and low affinity states are controlled by exposing the chelator to alternate cycles of blu and green/red light. We will exploit this optical control scheme in microscope-based studies to generate frequency and amplitude-defined waveforms of Ca2+ concentration with micron resolution in living cells. We will further show how optical control of the two states of a photochromic chelator can be used to generate defined Ca2+ oscillations in single cells, and between different cells in an epithelial colony. We will image the amplitude and frequency of Ca2+ waves in NBT-II epithelia, and test the hypothesis these waves are coincident with the activation of Ca2+-effector proteins. We will also test the hypothesis that these oscillatory signals are responsible for the waves of endosomes that are generated in HeLa cells following treatment with PDGF, and amplitude fluctuations in actomyosin contractility and waves of actin polymerization that are known to occur in many cells.